Perspective on the origin, resistance, and spread of the emerging human fungal pathogen Candida auris

What explains the origin, emergence, and persistence of Candida auris? Candida auris is a major emerging human fungal pathogen that was first reported in 2009 as an isolate from the ear canal of a patient in Japan [1]. Since this initial report, C. auris has grown to represent a “serious threat” in healthcare settings, as indicated by the United States Centers for Disease Control (CDC) and is now on the World Health Organization (WHO) fungal priority pathogen list, described as the “most wanted” critical pathogen [2,3]. However, many questions remain about the emergence, spread, and persistence of C. auris. In particular, the sudden, simultaneous, and independent worldwide emergence of 5 C. auris clades in completely separate geographical regions is a profound puzzle. One hypothesis suggests that C. auris was not detected until recently as a consequence of nonreliance on conventional phenotypic typing methods. Correct identification of C. auris is crucial for the adequate treatment and control of outbreaks and has been a frequent limitation. C. auris can be grown under similar conditions to those of other Candida species and single colonies can be obtained on conventional Sabouraud dextrose agar following 24 hours of incubation at 30 to 35 ̊C [4]. However, the ability of C. auris to grow at temperatures up to 42 ̊C differentiates it from other Candida species [5]. In clinical microbiology laboratories C. auris is frequently undetected, as 90% of isolates are misdiagnosed as Candida haemulonii, Candida famata, Candida guilliermondii, Candida lusitaniae, Candida parapsilosis, Candida sake, Rhodotorula glutinis, Candida duobushaemulonii, Candida catenulata, Candida tropicalis, or Saccharomyces cerevisiae, with commercial identification systems that utilize biochemical phenotyping [6–8]. C. auris appears white, pink, or purple on conventional CHROMagar Candida chromogenic medium (Becton-Dickinson, Rungis, France) but on CAN2 plates (bioMérieux, Capronne, France), colonies are initially white, but later appear as a light reddish pink color [5]. Recently, CHROMagar Candida Plus (Becton-Dickinson, Rungis, France) and HiCrome C. auris MDR selective agar (HiMedia, Mumbai, India) have been found to be highly specific and sensitive for the isolation and identification of C. auris after 36 to 48 hours of incubation [9–11]. FDA-approved methods such as MALDI-TOF mass spectrometry, combined with upto-date spectra databases, as well as the user-made MSI library (Paris, France) have also been used for the definitive identification of C. auris [12]. Finally, a combination of D1/D2 and ITS sequencing have proven to be the gold standard for C. auris identification [13]. While recent advances in fungal molecular diagnostics have improved C. auris detection and identification, the hypothesis that C. auris was not accurately detected is not alone sufficient to explain the PLOS PATHOGENS

sudden emergence of this species, since a reanalysis of Candida isolates between 1997 and 2016 detected only a small number of misidentified C. auris strains [14].
Another hypothesis suggests that extensive use of antifungals in the clinic and in agricultural settings could serve as a possible selective force in generating a transmission reservoir of antifungal-resistant C. auris strains; a similar hypothesis has been used to explain the emergence of azole-resistant Aspergillus fumigatus strains [15]. In support of this hypothesis, several C. auris strains, most of which were cross-resistant to medical and agricultural azoles, have been isolated from the surface of apples, although contamination from human handling cannot be excluded [16]. However, this hypothesis cannot explain the sudden worldwide emergence of C. auris belonging to 5 different clades, which suggests that other important contributing factors play a role.
The recently described "global warming emergence hypothesis" suggests that an increase in global warming led to the simultaneous emergence of thermal tolerant C. auris in different geographical locations [17]. Because C. auris is tolerant of temperature and salinity, Casadevall and colleagues have proposed that prior to being recognized as a human pathogenic species, C. auris existed as a plant saprophyte in specialized ecosystems, such as wetlands [18]. In support of this hypothesis, Arora and colleagues recently described the first environmental isolations of C. auris from a sandy beach and a salt marsh wetland in the Andaman Islands, India [19]. The isolation of 2 clonal strains, one of which exhibited slow growth at 37˚and 42˚C and was susceptible to antifungals and a second that grew well at 37˚and 42˚C, suggested a close association with the wild C. auris inhabiting the environment. These findings suggest that C. auris existed as a slow growing and drug-susceptible pathogen, which acquired thermal tolerance initially as a consequence of global warming and then developed drug resistance after its adaptation in humans [13]. In addition, the fact that C. auris colonizes colder body areas and is unable to grow in the absence of free oxygen may suggest an environmental origin. Acquisition of virulence traits in C. auris could also be explained by a combination of global warming and UV radiation that might have induced genetic mutations and/or epigenetic changes leading to improved adaptability for growth in different ecological niches [18,20]. Although the global warming hypothesis is well supported and may seem attractive, other factors, including global human migration, poor hygiene, and high population densities, should not be ignored and might also have contributed to the development of persistence and antifungal resistance in C. auris.

What are the molecular and genetic determinants of antifungal resistance in C. auris?
C. auris is best known for its strong resistance to a wide variety of antifungal therapies. Based on the tentative breakpoints proposed by the CDC, about 90%, 30%, and 2% to 10% of C. auris isolates are resistant to the major antifungal drugs fluconazole (FLU), amphotericin B (AMB), and echinocandins, respectively [21]. Overall, about 90% of C. auris strains have acquired resistance to at least 1 drug, 30% to 41% are resistant to 2 drugs and about 4% are resistant to all 3 antifungals [21]. It is also important to mention here that C. auris has been classified in 5 phylogenetically distinct clades based on whole-genome sequence data, with each clade differing from the other clades by >200,000 SNPs [22-25]. Table 1 describes the main characteristics of each clade. Recently, genomic and centromeric analyses have also demonstrated extensive rearrangements across all 7 chromosomes of C. auris [23,26,27]. The broad geographical distribution and genetic diversity among C. auris isolates belonging to different clades, as well as conflicting virulence reports, suggest that all clades need to be studied extensively and that the data from one clade cannot necessarily be extrapolated to others.
Azole resistance in C. auris has been mainly attributed to mutations in the ERG11 gene, which encodes the azole target lanosterol 14-α-demethylase (Table 2) [30,31]. Increased ERG11 copy number, largely observed in Clade III strains, may amplify the effects of these mutations. This finding also suggests clade-specific variation in azole resistance mechanisms [31]. Increased expression of CDR1, an ABC-transporter, has been shown to substantially contribute to C. auris clinical azole resistance and significantly increased expression of MDR1, encoding a MFS transporter, has been observed in FLU-resistant Clade III clinical C. auris strains ( In 2019, Ruiz and colleagues showed that the genome of C. auris undergoes substantial karyotypic reorganization under stress conditions [43]. The authors also hypothesized that as neither polyploid states nor sexual reproduction have been described in C. auris, it is highly likely that this species is not capable of generating genome diversity via aneuploidy or polyploidy. However, Burrack and colleagues have recently shown that C. auris acquires aneuploidy very rapidly in just 3 passages [44]. This aneuploidy can be easily detected in the population during in vitro evolution, in the presence and absence of FLU [44]. Another instance of rapid   . In addition, Narayanan and colleagues recently demonstrated a novel mechanism of antifungal resistance involving generation of a mitotically stable supernumerary chromosome, which leads to an increase in the copy number of a set of genes in C. auris [46]. Echinocandins inhibit the activity of 1,3-β-D-glucan synthase, an enzyme encoded by the FKS1 and FKS2 genes, which is important for synthesis of a primary fungal cell wall polymer. About 2% to 10% of clinical C. auris strains exhibit echinocandin resistance, which usually emerges during treatment [13]. The most commonly observed mutations associated with echinocandin resistance in C. auris are located in the FKS1 hot spot regions (Table 2), although their direct role in echinocandin susceptibility has not yet been assessed [39,48-56]. Interestingly, in a C. auris outbreak study, an isolate was found to be resistant to both echinocandins and 5-flucytosine (5-FC) [43]. Echinocandin resistance in this strain was associated with a serine to tyrosine amino acid substitution in the gene FKS1, while resistance to 5-FC was associated with a phenylalanine to isoleucine substitution in the gene FUR1 (Table 2) [49].

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AMB has broad-spectrum activity against pathogenic fungi and exerts its antifungal effect by directly binding to ergosterol. While AMB resistance is rare in fungi, there have been reports suggesting a higher prevalence (approximately 30%) of AMB resistance in clinical C. auris strains, based on tentative CDC breakpoints. Recent studies have shown that mutations in ERG6 (encoding C-24 sterol methyltransferase) are associated with C. auris AMB resistance (Table 2) [56,59]. In vitro exposure of an AMB-resistant C. auris strain to AMB also resulted in increased expression of ERG1, ERG2, ERG6, and ERG13 (Table 2) [23]. Wasi and colleagues analyzed changes in the transcript levels of C. auris ABC transporters by qRT-PCR after shortterm exposure to AMB and terbinafine [58]. Interestingly, the CDR6 ortholog CAUR_04233 exhibited greater than 8-fold higher expression compared to that of other ABC transporters in response to AMB. The CDR6 ortholog CAUR_04233 was also strongly up-regulated following terbinafine treatment (Table 2) [58]. However, because the majority of the AMB-resistant strains lack mutations in ergosterol biosynthesis pathway genes, alternative resistance mechanisms are likely. Escandón and colleagues identified SNPs in AMB-resistant isolates in a transcription factor similar to FLO8 in Candida albicans (Table 2) [57]. Although these findings do

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not provide definitive proof of involvement of these proteins in AMB resistance, they do provide a future direction for research into AMB resistance in C. auris. For most human fungal pathogens, such as C. albicans, the level of antifungal resistance remains low. However, clinical outcomes are poor, leading to high mortality rates due to treatment failure [14]. The immune status of the patient, along with other host factors and pharmacologic issues, including interaction of drug and the fungus, can directly or indirectly affect therapeutic responses [60]. This contradictory relationship between low clinical resistance and an overall clinical outcome of therapeutic failure could be attributed to antifungal tolerance [61]. Antifungal tolerance/trailing growth is usually characteristic of susceptible strains that tend to grow slowly at inhibitory drug concentrations [62,63]. Azole tolerance in C. auris is enhanced as mother cells age, requires the molecular chaperone HSP90, and has been associated with gene duplication as well as overexpression of ERG11 and CDR1 [33,47]. Based on these studies, C. auris shows increased tolerance to azoles and could be more tolerant to echinocandins as well [33,47]. It is pertinent to mention here that C. auris in vitro evolution experiments have used MIC measurements to define antifungal resistance. However, information about the range of drug tolerance in clinical strains and how tolerance changes over time is still lacking [37,45]. Because the degree of tolerance and resistance varies depending on the intrinsic allele diversity of each isolate and phenotypic heterogeneity, it is important to understand the contribution of the genetic background of the species [63][64][65]. Recently, Burrack and colleagues evolved a set of 17 clinical isolates of C. auris belonging to different clades to determine whether resistance is stable in the absence of drug, the frequency of drug tolerance, and how genetic background affects strain evolutionary outcome [44]. Interestingly, they found that drug tolerance can occur in C. auris and shows variation among clinical isolates similar to that observed for other Candida species. This study also suggested that antifungal tolerance acquired by C. auris over time could ultimately lead to resistance [44, 63,66].
Although antifungal resistance remains a key feature of most C. auris infections, relatively little is known about resistance mechanisms in C. auris compared to those of other human fungal pathogens and many important questions remain outstanding. How do resistance mechanisms differ among C. auris strains belonging to different genetic clades? Why is multidrug resistance more prevalent in C. auris compared to other pathogenic fungi? Are there completely novel mechanisms that make C. auris unique in its ability to tolerate antifungals? Future studies that address these questions are most likely to have the greatest impact on our understanding of C. auris drug resistance mechanisms.

How did C. auris prevail during the COVID-19 pandemic?
Since its first report in late 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for Coronavirus Disease 2019 (COVID-19), has rapidly spread, causing a global health emergency that resulted in the official announcement of a massive pandemic by WHO in 2020 [67,68]. COVID-19 has exacerbated several preexisting conditions, leading to an increase in bacterial, fungal, and viral coinfections and superinfections in hospitalized patients [69]. Fungal infections pose a greater challenge, as a consequence of high mortality rates, limited diagnostics, and increased antifungal resistance, and severe clinical COVID-19 further increases the risk of invasive fungal infections [69]. C. auris is one such fungal pathogen that causes outbreaks in COVID-19 ICUs and hospitals worldwide [69]. C. auris patients shed viable yeast cells continuously from their skin, leading to the contamination of hospital environments, especially ICUs. Both C. auris and SARS-CoV-2 have been found in the hospital setting, including on IV poles, bedrails, hospital floors, windows, and air conditioner ducts [69]. COVID-19 patients become potentially more susceptible to C. auris colonization and infection as they develop acute respiratory distress syndrome requiring ICU admission, mechanical ventilation, and/or extracorporeal membrane oxygenation [69]. Importantly, C. auris and SARS CoV-2 share several common risk factors, including chronic kidney disease, diabetes mellitus, as well as the administration of broad-spectrum antibiotics and systemic steroids.
While it is crucial to rapidly identify and isolate patients colonized with C. auris, the pandemic has made it challenging to do so, due to overburdened, resource-limited, and overwhelmed hospital settings. Unfortunately, the COVID-19 pandemic has provided ideal conditions for C. auris outbreaks in hospital ICUs. As a consequence, more effective antifungal therapies are in even greater demand.

How to strategize therapeutics: New drugs or drug combinations?
Based on frequency of resistance profiles and the available literature, the echinocandin drug micafungin has been recommended as the first-line treatment for C. auris infections in adults [84]. However, the cost and limited availability of this treatment in most countries is a major concern. AMB is also recommended as a first-line treatment for neonates and infants [84].
Given the challenges of resistance, novel antifungals are needed in order to effectively treat C. auris infections. Several new antifungal agents with potential therapeutic effects against C. auris have been developed that are undergoing Phase II or Phase III clinical trials. Ibrexafungerp is the first representative of a novel class of glucan synthase inhibitors, known as triterpenoids, and is the most promising. It possesses potent activity against C. auris strains, including echinocandin-resistant isolates, in vitro leading to cellular deformation and pore formation, as well as inhibition of cell division [85]. Rezafungin is a novel long-lasting echinocandin that exhibits potent activity against C. auris isolates both in vitro and in vivo in a neutropenic mouse model [85]. However, this drug shows reduced activity against echinocandin-resistant C. auris isolates. Another new antifungal compound, MYC-053 (sodium 5-[1-(3,5-dichloro-2-hydroxyphenyl) methylideneamino]-6-methyl-1,2,3,4-tetrahydro-2,4-pyrimidinedionate), synthesized by TGV-Therapeutics (Wilmington, DE), is a representative of a novel class of antifungal agents [86]. MYC-053 functions by simultaneously inhibiting both intracellular nucleic acid synthesis and targeting the synthesis of chitin, a key cell wall component [86]. This small molecule compound shows strong fungicidal activity against azole-and echinocandin-resistant isolates and has potential activity against biofilms [86]. Fosmanogepix is another novel antifungal that targets the enzyme Gwt1, which is necessary for localization of phosphatidylinositol-anchored proteins to the fungal cell wall [85]. This drug exhibits potent activity against resistant C. auris isolates and has also shown strong in vivo efficacy in a murine-invasive candidiasis model. Finally, VT-1598 is a new tetrazole that selectively inhibits fungal Cyp51A (lanosterol demethylase). In a neutropenic mouse model of C. auris infection, treatment with VT-1598 resulted in a significant increase in survival and a reduction in both brain and kidney fungal burden [85].
Several additional potential therapeutics for the treatment of C. auris infections have been identified through high-throughput screening of drug repurposing libraries, including the organoselenium compound ebselen, alexidine dihydrochloride, and the antiparasitic drugs miltefosine and iodoquinol [87]. Given the number of therapeutics currently under development and in the pipeline, several of which could potentially be used in combination with current therapies, the probability of successfully treating life-threatening C. auris infections is likely to be significantly improved in the future.